No abstract

Down-modulation of target molecules in tumor cells by small interfering (si) RNAs is a promising anti-cancer strategy. A major challenge of this approach is the loss of silencing activity of the siRNAs in vivo. Our study aimed at investigating the influence of the serum compartment on the anti-tumor activity of siRNA directed against Polo-like kinase 1 (Plk1), a mitosis-associated serine/threonine kinase. The data showed that siRNA-induced suppression of Plk1 expression effectively reduced the viable cell mass and increased apoptosis in several cancer cell lines. Preincubation of the siRNA in human serum led to shortening of the siRNA as well as loss of its Plk1 silencing and anti-tumor activity. This loss of activity was prevented by inhibition of RNAse A-like enzymes. These data indicate that the anti-neoplastic effect of siRNAs declines upon incubation in human serum. This loss of anti-neoplastic activity can be prevented by inhibition of their degradation by RNAse A-like enzymes. This may have important implications for the development of a human therapeutic application of siRNAs. ' 2007 Wiley-Liss, Inc.

Given the important role of cAMP in cell proliferation (1), we initially studied the effect of membrane-permeable Epac-selective and non-selective cAMP analogs on cell proliferation. We found that 8-pCPT-substituted cAMP analogs as well as their potential hydrolysis products strongly inhibit The transport of nucleosides into mammalian cells is accomplished by two types of transporters: concentrative nucleoside transporters, which use the Na ϩ gradient to actively transport nucleosides into the cells, and equilibrative nucleoside transporters (ENTs), which transport nucleosides in both directions and toward equal intra-and extracellular adenosine concentration (8, 9). ENTs play key roles in nucleoside and nucleobase uptake for salvage pathways of nucleotide synthesis and are also responsible for the cellular uptake of nucleoside analogues used in the treatment of cancers and viral diseases. By regulating the concentration of adenosine available to ubiquitously expressed cell surface adenosine receptors, nucleoside transporters appear to play important regulatory roles in many physiological * This work was supported by grants from the Deutsche Forschungsgemeinschaft (Grant Pi 258/7-4) and in part by grants from the University of Saarland (HOMFOR), the Wilhelm Sander foundation (Grant 2003.119.2), and the Marie Christine Held and Erika Hecker foundation.

Protein kinase C (PKC) is a key regulatory element in signal transduction and exerts its effects by catalyzing specific substrate phosphorylation. Today, P K C covers a family of 12 so far known phospholipid-dependent serine-threonine specific protein kinases. PKCdelta, the most thoroughly studied member of the socalled nPKC-subclass, is involved in diverse cellular processes, such as growth, differentiation, apoptosis and tumorigenesis. In order to understand the unique functions of PKCdelta, it is necessary to identify and characterize its specific substrates and interacting proteins. By using the yeast two-hybrid system we were able to identify a novel protein kinase termed DIK (for PKCDelta Interacting protein Kinase). A highly conserved protein kinase domain is present in the N-terminal moiety of DIK. Moreover, DIK exhibits 10 ankyrin-like repeats and a putative nuclear localization signal. Using a DIK-specific antibody expression of DIK can be detected in various cell lines. Recombinant DIK catalyzes autophosphorylation and phosphorylation of several common protein kinase substrate proteins. D I K is coimmunoprecipitated with PKCdelta from cell extracts.1548 SignaI transduction pathways invoked in the activation of sugar transport by stress in Clone 9 celb one of the early characteristic cellular responses to stressful conditions is an increase in the uptake and utilisation of glucose. The focus of our present study is transport stimulation resulting primerily h m activation of the GLUTl transporter isoform present at the cell surface of the rat liver cell line, Clone 9. Initial studies, using digital imaging microscopy of immwfhorescently-labelled plasma membrane lawns, provided a measuTe of cell surface GLUTl levels independent of transporter activity. Subjection of Clone 9 cells to osmotic and metabolic stress was found to stimulate sugar transport 3.6 f 0.2 (3) fold and 5.6 f 1.0 (3) fold, respectively, with only a small inCreaSe, 1.7 f 0.07 (3) fold, or no effect, respectively, on the abundance of plasma membrane GLUTl. These tindings suggest that most of the transport stimulation is due to traosporter activation i.e. an increase in the intrinsii activity of transporters at the cell s&.Interestingly, the cell surface transporters of non-stimulated cells not only had a reduced transporter activity but were also poorly labelled by the membrane-impermeable photoa%hity label Bio-LC-ATB-BMPA.The signal transduction pathways involved in these responses remain unclear. By using membrane permeable inhibitors we have, however, identified a possible candidate protein kinase signalling pathway for the response of Clone 9 cells to metabolic stress.Acknowledgements: We thank the BBSRC for support.